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1.
ACS Synth Biol ; 12(1): 153-163, 2023 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-36623275

RESUMO

Botulinum neurotoxin serotype A (BoNT/A) is a widely used cosmetic agent that also has diverse therapeutic applications; however, adverse antidrug immune responses and associated loss of efficacy have been reported in clinical uses. Here, we describe computational design and ultrahigh-throughput screening of a massive BoNT/A light-chain (BoNT/A-LC) library optimized for reduced T cell epitope content and thereby dampened immunogenicity. We developed a functional assay based on bacterial co-expression of BoNT/A-LC library members with a Förster resonance energy transfer (FRET) sensor for BoNT/A-LC enzymatic activity, and we employed high-speed fluorescence-activated cell sorting (FACS) to identify numerous computationally designed variants having wild-type-like enzyme kinetics. Many of these variants exhibited decreased immunogenicity in humanized HLA transgenic mice and manifested in vivo paralytic activity when incorporated into full-length toxin. One variant achieved near-wild-type paralytic potency and a 300% reduction in antidrug antibody response in vivo. Thus, we have achieved a striking level of BoNT/A-LC functional deimmunization by combining computational library design and ultrahigh-throughput screening. This strategy holds promise for deimmunizing other biologics with complex superstructures and mechanisms of action.


Assuntos
Anticorpos , Camundongos , Animais , Camundongos Transgênicos , Biblioteca Gênica , Domínios Proteicos
2.
Comput Struct Biotechnol J ; 20: 2169-2180, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35615020

RESUMO

The therapeutic efficacy of a protein binder largely depends on two factors: its binding site and its binding affinity. Advances in in vitro library display screening and next-generation sequencing have enabled accelerated development of strong binders, yet identifying their binding sites still remains a major challenge. The differentiation, or "binning", of binders into different groups that recognize distinct binding sites on their target is a promising approach that facilitates high-throughput screening of binders that may show different biological activity. Here we study the extent to which the information contained in the amino acid sequences comprising a set of target-specific binders can be leveraged to bin them, inferring functional equivalence of their binding regions, or paratopes, based directly on comparison of the sequences, their modeled structures, or their modeled interactions. Using a leucine-rich repeat binding scaffold known as a "repebody" as the source of diversity in recognition against interleukin-6 (IL-6), we show that the "Epibin" approach introduced here effectively utilized structural modelling and docking to extract specificity information encoded in the repebody amino acid sequences and thereby successfully recapitulate IL-6 binding competition observed in immunoassays. Furthermore, our computational binning provided a basis for designing in vitro mutagenesis experiments to pinpoint specificity-determining residues. Finally, we demonstrate that the Epibin approach can extend to antibodies, retrospectively comparing its predictions to results from antigen-specific antibody competition studies. The study thus demonstrates the utility of modeling structure and binding from the amino acid sequences of different binders against the same target, and paves the way for larger-scale binning and analysis of entire repertoires.

3.
Nat Commun ; 12(1): 6947, 2021 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-34845212

RESUMO

Each year vast international resources are wasted on irreproducible research. The scientific community has been slow to adopt standard software engineering practices, despite the increases in high-dimensional data, complexities of workflows, and computational environments. Here we show how scientific software applications can be created in a reproducible manner when simple design goals for reproducibility are met. We describe the implementation of a test server framework and 40 scientific benchmarks, covering numerous applications in Rosetta bio-macromolecular modeling. High performance computing cluster integration allows these benchmarks to run continuously and automatically. Detailed protocol captures are useful for developers and users of Rosetta and other macromolecular modeling tools. The framework and design concepts presented here are valuable for developers and users of any type of scientific software and for the scientific community to create reproducible methods. Specific examples highlight the utility of this framework, and the comprehensive documentation illustrates the ease of adding new tests in a matter of hours.


Assuntos
Substâncias Macromoleculares/química , Simulação de Acoplamento Molecular , Proteínas/química , Software/normas , Benchmarking , Sítios de Ligação , Humanos , Ligantes , Substâncias Macromoleculares/metabolismo , Ligação Proteica , Proteínas/metabolismo , Reprodutibilidade dos Testes
4.
Retrovirology ; 18(1): 35, 2021 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-34717659

RESUMO

BACKGROUND: The critical role of antibody Fc-mediated effector functions in immune defense has been widely reported in various viral infections. These effector functions confer cellular responses through engagement with innate immune cells. The precise mechanism(s) by which immunoglobulin G (IgG) Fc domain and cognate receptors may afford protection are poorly understood, however, in the context of HIV/SHIV infections. Many different in vitro assays have been developed and utilized to measure effector functions, but the extent to which these assays capture distinct antibody activities has not been fully elucidated. RESULTS: In this study, six Fc-mediated effector function assays and two biophysical antibody profiling assays were performed on a common set of samples from HIV-1 infected and vaccinated subjects. Biophysical antibody profiles supported robust prediction of diverse IgG effector functions across distinct Fc-mediated effector function assays. While a number of assays showed correlated activities, supervised machine learning models indicated unique antibody features as primary contributing factors to the associated effector functions. Additional experiments established the mechanistic relevance of relationships discovered using this unbiased approach. CONCLUSIONS: In sum, this study provides better resolution on the diversity and complexity of effector function assays, offering a clearer perspective into this family of antibody mechanisms of action to inform future HIV-1 treatment and vaccination strategies.


Assuntos
Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/química , Imunoglobulina G/imunologia , Infecções por HIV/imunologia , Humanos
5.
PLoS Comput Biol ; 17(10): e1009470, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34613971

RESUMO

Lectin-glycan interactions facilitate inter- and intracellular communication in many processes including protein trafficking, host-pathogen recognition, and tumorigenesis promotion. Specific recognition of glycans by lectins is also the basis for a wide range of applications in areas including glycobiology research, cancer screening, and antiviral therapeutics. To provide a better understanding of the determinants of lectin-glycan interaction specificity and support such applications, this study comprehensively investigates specificity-conferring features of all available lectin-glycan complex structures. Systematic characterization, comparison, and predictive modeling of a set of 221 complementary physicochemical and geometric features representing these interactions highlighted specificity-conferring features with potential mechanistic insight. Univariable comparative analyses with weighted Wilcoxon-Mann-Whitney tests revealed strong statistical associations between binding site features and specificity that are conserved across unrelated lectin binding sites. Multivariable modeling with random forests demonstrated the utility of these features for predicting the identity of bound glycans based on generalized patterns learned from non-homologous lectins. These analyses revealed global determinants of lectin specificity, such as sialic acid glycan recognition in deep, concave binding sites enriched for positively charged residues, in contrast to high mannose glycan recognition in fairly shallow but well-defined pockets enriched for non-polar residues. Focused fine specificity analysis of hemagglutinin interactions with human-like and avian-like glycans uncovered features representing both known and novel mutations related to shifts in influenza tropism from avian to human tissues. As the approach presented here relies on co-crystallized lectin-glycan pairs for studying specificity, it is limited in its inferences by the quantity, quality, and diversity of the structural data available. Regardless, the systematic characterization of lectin binding sites presented here provides a novel approach to studying lectin specificity and is a step towards confidently predicting new lectin-glycan interactions.


Assuntos
Lectinas , Polissacarídeos , Aminoácidos/química , Sítios de Ligação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Lectinas/química , Lectinas/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Ligação Proteica
6.
PLoS Comput Biol ; 17(4): e1008889, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33793553

RESUMO

Bacteria utilize a wide variety of endogenous cell wall hydrolases, or autolysins, to remodel their cell walls during processes including cell division, biofilm formation, and programmed death. We here systematically investigate the composition of these enzymes in order to gain insights into their associated biological processes, potential ways to disrupt them via chemotherapeutics, and strategies by which they might be leveraged as recombinant antibacterial biotherapies. To do so, we developed LEDGOs (lytic enzyme domains grouped by organism), a pipeline to create and analyze databases of autolytic enzyme sequences, constituent domain annotations, and architectural patterns of multi-domain enzymes that integrate peptidoglycan binding and degrading functions. We applied LEDGOs to eight pathogenic bacteria, gram negatives Acinetobacter baumannii, Klebsiella pneumoniae, Neisseria gonorrhoeae, and Pseudomonas aeruginosa; and gram positives Clostridioides difficile, Enterococcus faecium, Staphylococcus aureus, and Streptococcus pneumoniae. Our analysis of the autolytic enzyme repertoires of these pathogens reveals commonalities and differences in their key domain building blocks and architectures, including correlations and preferred orders among domains in multi-domain enzymes, repetitions of homologous binding domains with potentially complementarity recognition modalities, and sequence similarity patterns indicative of potential divergence of functional specificity among related domains. We have further identified a variety of unannotated sequence regions within the lytic enzymes that may themselves contain new domains with important functions.


Assuntos
Proteínas de Bactérias/metabolismo , Biologia Computacional/métodos , Bases de Dados de Proteínas , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Positivas/enzimologia , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Antibacterianos/metabolismo , Antibacterianos/farmacologia , N-Acetil-Muramil-L-Alanina Amidase/farmacologia
7.
J Chem Inf Model ; 61(5): 2368-2382, 2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-33900750

RESUMO

As non-"self" macromolecules, biotherapeutics can trigger an immune response that can reduce drug efficacy, require patients to be taken off therapy, or even cause life-threatening reactions. To enable the flexible and facile design of protein biotherapeutics while reducing the prevalence of T-cell epitopes that drive immune recognition, we have integrated into the Rosetta protein design suite a new scoring term that allows design protocols to account for predicted or experimentally identified epitopes in the optimized objective function. This flexible scoring term can be used in any Rosetta design trajectory, can be targeted to specific regions of a protein, and can be readily extended to work with a variety of epitope predictors. By performing extensive design runs with varied design parameter choices for three case study proteins as well as a larger diverse benchmark, we show that the incorporation of this scoring term enables the effective exploration of an alternative, deimmunized sequence space to discover diverse proteins that are potentially highly deimmunized while retaining physical and chemical qualities similar to those yielded by equivalent nondeimmunizing sequence design protocols.


Assuntos
Biologia Computacional , Engenharia de Proteínas , Epitopos de Linfócito T , Humanos , Proteínas/genética
8.
Biotechnol Bioeng ; 118(7): 2482-2492, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33748952

RESUMO

Clostridioides difficile is the single most deadly bacterial pathogen in the United States, and its global prevalence and outsized health impacts underscore the need for more effective therapeutic options. Towards this goal, a novel group of modified peptidoglycan hydrolases with significant in vitro bactericidal activity have emerged as potential candidates for treating C. difficile infections (CDI). To date, discovery and development efforts directed at these CDI-specific lysins have been limited, and in particular there has been no systematic comparison of known or newly discovered lysin candidates. Here, we detail bioinformatics-driven discovery of six new anti-C. difficile lysins belonging to the amidase-3 family of enzymes, and we describe experimental comparison of their respective catalytic domains (CATs) with highly active CATs from the literature. Our quantitative analyses include metrics for expression level, inherent antibacterial activity, breadth of strain selectivity, killing of germinating spores, and structural and functional measures of thermal stability. Importantly, prior studies have not examined stability as a performance metric, and our results show that the panel of eight enzymes possess widely variable thermal denaturation temperatures and resistance to heat inactivation, including some enzymes that exhibit marginal stability at body temperature. Ultimately, no single enzyme dominated with respect to all performance measures, suggesting the need for a balanced assessment of lysin properties during efforts to find, engineer, and develop candidates with true clinical potential.


Assuntos
Proteínas de Bactérias , Clostridioides difficile , Biologia Computacional , N-Acetil-Muramil-L-Alanina Amidase , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Clostridioides difficile/enzimologia , Clostridioides difficile/genética , Humanos , N-Acetil-Muramil-L-Alanina Amidase/química , N-Acetil-Muramil-L-Alanina Amidase/genética , Domínios Proteicos
9.
Bioinformatics ; 37(17): 2580-2588, 2021 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-33693581

RESUMO

MOTIVATION: Protein-protein interactions drive wide-ranging molecular processes, and characterizing at the atomic level how proteins interact (beyond just the fact that they interact) can provide key insights into understanding and controlling this machinery. Unfortunately, experimental determination of three-dimensional protein complex structures remains difficult and does not scale to the increasingly large sets of proteins whose interactions are of interest. Computational methods are thus required to meet the demands of large-scale, high-throughput prediction of how proteins interact, but unfortunately, both physical modeling and machine learning methods suffer from poor precision and/or recall. RESULTS: In order to improve performance in predicting protein interaction interfaces, we leverage the best properties of both data- and physics-driven methods to develop a unified Geometric Deep Neural Network, 'PInet' (Protein Interface Network). PInet consumes pairs of point clouds encoding the structures of two partner proteins, in order to predict their structural regions mediating interaction. To make such predictions, PInet learns and utilizes models capturing both geometrical and physicochemical molecular surface complementarity. In application to a set of benchmarks, PInet simultaneously predicts the interface regions on both interacting proteins, achieving performance equivalent to or even much better than the state-of-the-art predictor for each dataset. Furthermore, since PInet is based on joint segmentation of a representation of a protein surfaces, its predictions are meaningful in terms of the underlying physical complementarity driving molecular recognition. AVAILABILITY AND IMPLEMENTATION: PInet scripts and models are available at https://github.com/FTD007/PInet. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

10.
Vaccine ; 38(46): 7239-7245, 2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-33004239

RESUMO

BACKGROUND: SRL172 prevented disease due to Mycobacterium tuberculosis in a Phase 3 trial. DAR-901 represents a scalable manufacturing process for SRL172. We sought to determine if DAR-901 would prevent infection with M. tuberculosis among BCG-primed adolescents age 13-15 years in Tanzania. METHODS: Adolescents with a negative T- SPOT.TBR interferon gamma release assay (IGRA) were randomized 1:1 to three intradermal injections of DAR-901 or saline placebo at 0, 2 and 4 months. Repeat IGRAs were performed at 2 months, and at 1, 2, and 3 years. The primary efficacy outcome was time to new TB infection (IGRA conversion to positive); the secondary outcome was time to persistent TB infection (IGRA conversion with repeat positive IGRA). RESULTS: Among 936 participants screened 667 were eligible and randomized to their first dose of vaccine or placebo (safety cohort). At 2 months, 625 participants remained IGRA-negative and were scheduled for the additional two doses (efficacy cohort). DAR-901 was safe and well-tolerated. One DAR-901 recipient developed a vaccine site abscess. Neither the primary nor secondary endpoints differed between the two treatment arms (p = 0.90 and p = 0.20, respectively). DAR-901 IGRA converters had median responses to ESAT-6 of 50.1 spot-forming cells (SFCs) vs. 19.6 SFCs in placebo IGRA converters (p = 0.03). CONCLUSIONS: A three-dose series of 1 mg DAR-901 was safe and well-tolerated but did not prevent initial or persistent IGRA conversion. DAR-901 recipients with IGRA conversion demonstrated enhanced immune responses to ESAT-6. Since protection against disease may require different immunologic responses than protection against infection a trial of DAR-901 to prevent TB disease is warranted. TRIAL REGISTRATION: The trial is registered at ClinicalTrials.gov as NCT02712424.


Assuntos
Mycobacterium tuberculosis , Tuberculose , Adolescente , Vacina BCG , Humanos , Testes de Liberação de Interferon-gama , Tanzânia , Teste Tuberculínico , Tuberculose/prevenção & controle
11.
Immunohorizons ; 4(10): 597-607, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33037097

RESUMO

The NKG2D ligand MHC class I chain-related protein A (MICA) is expressed on many varieties of malignant cells but is absent from most normal tissues, and thus represents a potential target for chimeric Ag receptor (CAR) T cell-based therapeutics. However, there are more than 100 alleles of MICA, so the ability to target a conserved site is needed for a therapy to be used in most patients. In this study, we describe a fully human anti-MICA CAR created by fusing the single-chain fragment variable B2 to the full length DAP10 protein and the traditional CD3ζ signaling domain. Human T cells expressing the B2 CAR killed MICA-positive tumor cells, produced IFN-γ when in contact with MICA-positive tumor cells or plate-bound MICA protein, and inhibited PANC-1 growth in a mouse xenograft model. To localize B2's epitope on MICA, we used novel computational methods to model potential binding modes and to design mutational variants of MICA testing these hypotheses. Flow cytometry using a commercial anti-MICA/MICB Ab indicated that the variant proteins were expressed at high levels on transduced P815 cell lines. One variant protein (R38S/K40T/K57E) showed reduced staining with a B2-IgG1 fusion protein compared with controls and did not induce IFN-γ production by human T cells expressing the B2 CAR. These results show antitumor activity of MICA-specific CAR T cells and indicate an essential role for a conserved site in the exposed loop involving aa 38-57 of MICA. This study describes a novel MICA-specific CAR and discusses its potential use as a cancer therapeutic.


Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Animais , Linhagem Celular Tumoral , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Transdução de Sinais , Ensaios Antitumorais Modelo de Xenoenxerto
12.
PLoS Comput Biol ; 16(8): e1008150, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866140

RESUMO

Precise binding mode identification and subsequent affinity improvement without structure determination remain a challenge in the development of therapeutic proteins. However, relevant experimental techniques are generally quite costly, and purely computational methods have been unreliable. Here, we show that integrated computational and experimental epitope localization followed by full-atom energy minimization can yield an accurate complex model structure which ultimately enables effective affinity improvement and redesign of binding specificity. As proof-of-concept, we used a leucine-rich repeat (LRR) protein binder, called a repebody (Rb), that specifically recognizes human IgG1 (hIgG1). We performed computationally-guided identification of the Rb:hIgG1 binding mode and leveraged the resulting model to reengineer the Rb so as to significantly increase its binding affinity for hIgG1 as well as redesign its specificity toward multiple IgGs from other species. Experimental structure determination verified that our Rb:hIgG1 model closely matched the co-crystal structure. Using a benchmark of other LRR protein complexes, we further demonstrated that the present approach may be broadly applicable to proteins undergoing relatively small conformational changes upon target binding.


Assuntos
Proteínas/química , Humanos , Proteínas de Repetições Ricas em Leucina , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Proteínas/metabolismo
13.
Sci Adv ; 6(36)2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32917596

RESUMO

There is a critical need for novel therapies to treat methicillin-resistant Staphylococcus aureus (MRSA) and other drug-resistant pathogens, and lysins are among the vanguard of innovative antibiotics under development. Unfortunately, lysins' own microbial origins can elicit detrimental antidrug antibodies (ADAs) that undermine efficacy and threaten patient safety. To create an enhanced anti-MRSA lysin, a novel variant of lysostaphin was engineered by T cell epitope deletion. This "deimmunized" lysostaphin dampened human T cell activation, mitigated ADA responses in human HLA transgenic mice, and enabled safe and efficacious repeated dosing during a 6-week longitudinal infection study. Furthermore, the deimmunized lysostaphin evaded established anti-wild-type immunity, thereby providing significant anti-MRSA protection for animals that were immune experienced to the wild-type enzyme. Last, the enzyme synergized with daptomycin to clear a stringent model of MRSA endocarditis. By mitigating T cell-driven antidrug immunity, deimmunized lysostaphin may enable safe, repeated dosing to treat refractory MRSA infections.


Assuntos
Lisostafina , Staphylococcus aureus Resistente à Meticilina , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Humanos , Lisostafina/farmacologia , Lisostafina/uso terapêutico , Camundongos , Camundongos Transgênicos
14.
Molecules ; 25(16)2020 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-32796656

RESUMO

Vaccines and immunotherapies depend on the ability of antibodies to sensitively and specifically recognize particular antigens and specific epitopes on those antigens. As such, detailed characterization of antibody-antigen binding provides important information to guide development. Due to the time and expense required, high-resolution structural characterization techniques are typically used sparingly and late in a development process. Here, we show that antibody-antigen binding can be characterized early in a process for whole panels of antibodies by combining experimental and computational analyses of competition between monoclonal antibodies for binding to an antigen. Experimental "epitope binning" of monoclonal antibodies uses high-throughput surface plasmon resonance to reveal which antibodies compete, while a new complementary computational analysis that we call "dock binning" evaluates antibody-antigen docking models to identify why and where they might compete, in terms of possible binding sites on the antigen. Experimental and computational characterization of the identified antigenic hotspots then enables the refinement of the competitors and their associated epitope binding regions on the antigen. While not performed at atomic resolution, this approach allows for the group-level identification of functionally related monoclonal antibodies (i.e., communities) and identification of their general binding regions on the antigen. By leveraging extensive epitope characterization data that can be readily generated both experimentally and computationally, researchers can gain broad insights into the basis for antibody-antigen recognition in wide-ranging vaccine and immunotherapy discovery and development programs.


Assuntos
Anticorpos Monoclonais/imunologia , Antígenos Virais/imunologia , Mapeamento de Epitopos/métodos , Epitopos/imunologia , Vacinas contra o Vírus do Herpes Simples/imunologia , Herpesvirus Humano 1/imunologia , Proteínas do Envelope Viral/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Antígenos Virais/metabolismo , Ligação Competitiva , Vacinas contra o Vírus do Herpes Simples/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Conformação Proteica , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo
15.
ACS Comb Sci ; 22(9): 446-456, 2020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-32574486

RESUMO

Mutagenesis of surface-exposed residues, or "resurfacing", is a protein engineering strategy that can be utilized to disrupt antibody recognition or modulate the capacity of a protein to elicit antibody responses. We apply resurfacing to engineer Dengue virus envelope protein domain III (DENV DIII) antigens with the goal of focusing humoral recognition on epitopes of interest by selective ablation of irrelevant and undesired epitopes. Cross-reactive but non-neutralizing antibodies have the potential to enhance Dengue virus (DENV) infection by a process called antibody-dependent enhancement, thought to be associated with severe secondary heterotypic infection. Thus, a focus on epitopes associated with broadly neutralizing antibodies is important both for understanding human antibody responses against DENV and for the development of a successful DENV vaccine. To engineer DENV DIII antigens focusing on the AG strand epitope associated with broadly neutralizing antibody responses, we generated yeast surface display libraries of DENV2 DIII where the AB loop (associated with cross-reactive but non-neutralizing antibody responses) and FG loop (associated with serotype-specific antibody responses) were mutagenized to allow for all possible amino acid substitutions. Loop variants that maintained the AG strand epitope and simultaneously disrupted the AB and FG loop epitopes exhibited high and diverse mutational loads that were amenable to loop exchange and transplantation into a DENV4 DIII background. Thus, several loop variants fulfill this antigenicity criteria regardless of serotype context. The resulting resurfaced DIII antigens may be utilized as AG strand epitope-focusing probes or immunogen candidates.


Assuntos
Anticorpos Antivirais/química , Antígenos Virais/química , Vírus da Dengue/química , Epitopos/química , Proteínas do Envelope Viral/química , Anticorpos Antivirais/imunologia , Reações Antígeno-Anticorpo , Antígenos Virais/imunologia , Vírus da Dengue/imunologia , Epitopos/imunologia , Proteínas do Envelope Viral/imunologia
16.
Bioinformatics ; 36(13): 3996-4003, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32321157

RESUMO

MOTIVATION: Understanding how antibodies specifically interact with their antigens can enable better drug and vaccine design, as well as provide insights into natural immunity. Experimental structural characterization can detail the 'ground truth' of antibody-antigen interactions, but computational methods are required to efficiently scale to large-scale studies. To increase prediction accuracy as well as to provide a means to gain new biological insights into these interactions, we have developed a unified deep learning-based framework to predict binding interfaces on both antibodies and antigens. RESULTS: Our framework leverages three key aspects of antibody-antigen interactions to learn predictive structural representations: (i) since interfaces are formed from multiple residues in spatial proximity, we employ graph convolutions to aggregate properties across local regions in a protein; (ii) since interactions are specific between antibody-antigen pairs, we employ an attention layer to explicitly encode the context of the partner; (iii) since more data are available for general protein-protein interactions, we employ transfer learning to leverage this data as a prior for the specific case of antibody-antigen interactions. We show that this single framework achieves state-of-the-art performance at predicting binding interfaces on both antibodies and antigens, and that each of its three aspects drives additional improvement in the performance. We further show that the attention layer not only improves performance, but also provides a biologically interpretable perspective into the mode of interaction. AVAILABILITY AND IMPLEMENTATION: The source code is freely available on github at https://github.com/vamships/PECAN.git.


Assuntos
Anticorpos , Software , Transdução de Sinais
17.
Vaccine ; 38(18): 3436-3446, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32192810

RESUMO

Antibodies against the HIV-1 V1V2 loops were the only correlate of reduced infection risk in the RV144 vaccine trial, highlighting the V1V2 loops as promising targets for vaccine design. The V1V2 loops are structurally plastic, exhibiting either an α-helix-coil or ß-strand conformation. V1V2-specific antibodies may thus recognize distinct conformations, and an antibody's conformational specificity can be an important determinant of breadth and function. Restricting V1V2 conformational plasticity in an immunogen may thus provide control over the conformational specificity and quality of a vaccine-elicited antibody response. Previously, we identified a V1V2 sequence variant (K155M) that results in enhanced recognition by cross-reactive antibodies recognizing the ß-strand conformation. Here, we relate V1V2 antigenicity to immunogenicity by comparing the immunogenicity profiles of wildtype and K155M immunogens in two mouse models. In one model, immunization with gp70 V1V2 K155M but not wildtype elicited antibody responses that were cross-reactive to a panel of heterologous gp120 and gp140 antigens. In a second model, we compared the effect of K155M on immunogenicity in the context of gp70 V1V2, gD V1V2 and gp120, examining the effects of scaffold, epitope-focusing and immunization regimen. K155M variants, especially in the context of a gp120 immunogen, resulted in more robust, durable and cross-reactive antibody responses than wildtype immunogens. Restriction of the ß-stranded V1V2 conformation in K155M immunogens may thus be associated with the induction of cross-reactive antibody responses thought to be required of a protective HIV-1 vaccine.


Assuntos
Vacinas contra a AIDS , Anticorpos Anti-HIV , Infecções por HIV , Animais , Anticorpos Neutralizantes , Formação de Anticorpos , Proteína gp120 do Envelope de HIV/genética , HIV-1/genética , Camundongos
18.
Mol Syst Biol ; 15(5): e8747, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-31048360

RESUMO

Characterizing the antigen-binding and innate immune-recruiting properties of the humoral response offers the chance to obtain deeper insights into mechanisms of protection than revealed by measuring only overall antibody titer. Here, a high-throughput, multiplexed Fab-Fc Array was employed to profile rhesus macaques vaccinated with a gp120-CD4 fusion protein in combination with different genetically encoded adjuvants, and subsequently subjected to multiple heterologous simian immunodeficiency virus (SIV) challenges. Systems analyses modeling protection and adjuvant differences using Fab-Fc Array measurements revealed a set of correlates yielding strong and robust predictive performance, while models based on measurements of response magnitude alone exhibited significantly inferior performance. At the same time, rendering Fab-Fc measurements mathematically independent of titer had relatively little impact on predictive performance. Similar analyses for a distinct SIV vaccine study also showed that Fab-Fc measurements performed significantly better than titer. These results suggest that predictive modeling with measurements of antibody properties can provide detailed correlates with robust predictive power, suggest directions for vaccine improvement, and potentially enable discovery of mechanistic associations.


Assuntos
Anticorpos Antivirais/imunologia , Fragmentos de Imunoglobulinas/imunologia , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vírus da Imunodeficiência Símia/imunologia , Animais , Linfócitos T CD4-Positivos/citologia , Humanos , Imunidade Humoral , Imunoglobulina G/imunologia , Macaca mulatta , Glicoproteínas de Membrana/imunologia , Análise Multivariada , Proteínas do Envelope Viral/imunologia
19.
BMC Bioinformatics ; 20(1): 241, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31092185

RESUMO

BACKGROUND: Repertoire sequencing is enabling deep explorations into the cellular immune response, including the characterization of commonalities and differences among T cell receptor (TCR) repertoires from different individuals, pathologies, and antigen specificities. In seeking to understand the generality of patterns observed in different groups of TCRs, it is necessary to balance how well each pattern represents the diversity among TCRs from one group (sensitivity) vs. how many TCRs from other groups it also represents (specificity). The variable complementarity determining regions (CDRs), particularly the third CDRs (CDR3s) interact with major histocompatibility complex (MHC)-presented epitopes from putative antigens, and thus encode the determinants of recognition. RESULTS: We here systematically characterize the predictive power that can be obtained from CDR3 sequences, using representative, readily interpretable methods for evaluating CDR sequence similarity and then clustering and classifying sequences based on similarity. An initial analysis of CDR3s of known structure, clustered by structural similarity, helps calibrate the limits of sequence diversity among CDRs that might have a common mode of interaction with presented epitopes. Subsequent analyses demonstrate that this same range of sequence similarity strikes a favorable specificity/sensitivity balance in distinguishing twins from non-twins based on overall CDR3 repertoires, classifying CDR3 repertoires by antigen specificity, and distinguishing general pathologies. CONCLUSION: We conclude that within a fairly broad range of sequence similarity, matching CDR3 sequences are likely to share specificities.


Assuntos
Regiões Determinantes de Complementaridade/química , Receptores de Antígenos de Linfócitos T/química , Homologia de Sequência de Aminoácidos , Motivos de Aminoácidos , Sequência de Aminoácidos , Epitopos/química , Humanos , Complexo Principal de Histocompatibilidade , Peptídeos/química , Gêmeos
20.
IEEE/ACM Trans Comput Biol Bioinform ; 16(4): 1143-1153, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30040654

RESUMO

In order to increase the hit rate of discovering diverse, beneficial protein variants via high-throughput screening, we have developed a computational method to optimize combinatorial mutagenesis libraries for overall enrichment in two distinct properties of interest. Given scoring functions for evaluating individual variants, POCoM (Pareto Optimal Combinatorial Mutagenesis) scores entire libraries in terms of averages over their constituent members, and designs optimal libraries as sets of mutations whose combinations make the best trade-offs between average scores. This represents the first general-purpose method to directly design combinatorial libraries for multiple objectives characterizing their constituent members. Despite being rigorous in mapping out the Pareto frontier, it is also very fast even for very large libraries (e.g., designing 30 mutation, billion-member libraries in only hours). We here instantiate POCoM with scores based on a target's protein structure and its homologs' sequences, enabling the design of libraries containing variants balancing these two important yet quite different types of information. We demonstrate POCoM's generality and power in case study applications to green fluorescent protein, cytochrome P450, and ß-lactamase. Analysis of the POCoM library designs provides insights into the trade-offs between structure- and sequence-based scores, as well as the impacts of experimental constraints on library designs. POCoM libraries incorporate mutations that have previously been found favorable experimentally, while diversifying the contexts in which these mutations are situated and maintaining overall variant quality.


Assuntos
Biologia Computacional/métodos , Biblioteca Gênica , Mutagênese , Algoritmos , Sistema Enzimático do Citocromo P-450/genética , Proteínas de Fluorescência Verde/metabolismo , Modelos Moleculares , Mutação , Oligonucleotídeos/genética , Linguagens de Programação , Engenharia de Proteínas/métodos , Proteínas/genética , Software , beta-Lactamases/genética
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